The dependence of the rate of RNase A catalyzed hydrolysis of ribosomes and rRNA on the concentration of magnesium and ammonium ions

The introduction of non-natural amino acid residues or modules into proteins provides a new means to explore the basis for conformational stability, folding/unfolding behavior, or biological function. We exploited intein-mediated protein ligation to produce a semisynthetic ribonuclease A. Of the 124 residues of RNase A, residues 1–94 were linked to an intein. After expression of the fusion protein and thiol-induced cleavage, the RNase A(1–94) fragment possessed a C-terminal thioester. A peptide identical to the C-terminal residues 95–124 of RNase A (with residue 95 being cysteine) was successfully ligated to that thioester thereby reconstituting full-length wild-type RNase A. In mass spectrometry, this semisynthetic RNase A proved to be undistinguishable from the control protein, namely recombinant wild-type RNase A. Recombinant wild-type RNase A was obtained by expression of RNase A(1–124)–intein fusion protein followed by thiol-induced cleavage and hydrolysis of the thioester. Both proteins showed thermal stabilities (Tm) and catalytic activities comparable to the wild-type enzyme, indicating that both proteins folded properly. These results might serve as basis for the semisynthesis of RNase A variants containing non-natural modules in the aforementioned peptide.

Download Full-text

The hydrolysis of ammonium ions in sea water-experimental confirmation of predicted constants at one atmosphere pressure

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s0025315400041436 ◽

1978 ◽

Vol 58 (3) ◽

pp. 781-786 ◽

Cited By ~ 22

Author(s):

M. Whitfield

Keyword(s):

Sea Water ◽

Calculated Data ◽

Experimental Confirmation ◽

Ammonia Toxicity ◽

Experimental Measurements ◽

Ammonium Ions ◽

Atmosphere Pressure ◽

Hydrolysis Of

Recent experimental measurements have confirmed the accuracy of earlier calculations of the extent of hydrolysis of ammonium ions in sea water over a range of temperature and salinity. Tables of the percentage of un-ionized ammonia in sea water compiled from the calculated data can therefore be used with confidence in assessing ammonia toxicity in mariculture systems.

Download Full-text

Model studies concerning the first step in the hydrolysis of ribonucleic acids by RNase A

The Journal of Organic Chemistry ◽

10.1021/jo00329a008 ◽

1981 ◽

Vol 46 (16) ◽

pp. 3189-3193 ◽

Cited By ~ 4

Author(s):

Dirk Van Aken ◽

Luud M. C. Paulissen ◽

Henk M. Buck

Keyword(s):

Rnase A ◽

Ribonucleic Acids ◽

Model Studies ◽

Hydrolysis Of

Download Full-text

The Hydrolysis of Ammonium Ions in Sea Water-a Theoretical Study

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s002531540002275x ◽

1974 ◽

Vol 54 (3) ◽

pp. 565-580 ◽

Cited By ~ 123

Author(s):

M. Whitfield

Keyword(s):

Sea Water ◽

Practical Importance ◽

Lipid Solubility ◽

Ammonium Ions ◽

Theoretical Approaches ◽

High Lipid ◽

Ionic Equilibria ◽

Good Account ◽

High Lipid Solubility ◽

Hydrolysis Of

A number of procedures are now available for calculating the effects of ionic inter-actions on the behaviour of solutions as complex as sea water (Whitfield, 1973 a). These procedures are able to give a good account of the properties of the major electrolyte components (Leyendekkers, 1973 a; Robinson & Wood, 1972; Whitfield, 19736) and of the colligative properties of sea water (Robinson & Wood, 1972; Whitfield, 1973 c, d). However, greatest interest centres around the possibility of predicting the effects of these major components on the multitude of ionic equilibria that influence the properties of the less abundant constituents that are of greater biological and geological importance. It is here that the newer approaches to marine chemistry are weakest because suitable thermodynamic data are lacking. One system of practical importance that can be studied using a variety of theoretical approaches is the acid ionization of ammonium ions represented by the equation The toxicity of ammonium salts to freshwater life has been shown to be strongly dependent on the pH in a manner that is consistent with un-ionized ammonia (NH3) being the most lethal fraction (see, for example, Wuhrmann & Woker, 1948; Downing & Merkens, 1955; Lloyd & Herbert, i960; Hemens, 1966; and Brown, 1968). These papers and many others have been thoroughly reviewed (EIFAC Technical Paper no. 11, 1970, Kemp, Abrams & Overbeck, 1971). The free base (NH3) has a relatively high lipid solubility because it carries no charge and is therefore able to diffuse quite readily across cell membranes (Fromm & Gillette, 1968).

Download Full-text